Download The mapping of linear B-cell epitope regions in desmoglein 1 and 3

Journal of Peptide Science
The mapping of linear B-cell epitope regions in desmoglein
1 and 3 proteins: Recognition of immobilized peptides by
pemphigus patients’ serum autoantibodies
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Journal of Peptide Science
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Szabados, Hajnalka; Hungarian Academy of Sciences, Eötvös Loránd
University, Research Group of Peptide Chemistry
Bõsze, Szilvia; Hungarian Academy of Sciences, Eötvös Loránd University,
Research Group of Peptide Chemistry
Silló, Pálma; Semmelweis University, Department of Dermatology,
Venereology and Dermato-Oncology
Hudecz, Ferenc; Hungarian Academy of Sciences, Eötvös Loránd
University, Research Group of Peptide Chemistry; Eötvös Loránd
University, Institute of Chemistry
Kárpáti, Sarolta; Semmelweis University, Department of Dermatology,
Venereology and Dermato-Oncology
Uray, Katalin; Hungarian Academy of Sciences, Eötvös Loránd University,
Research Group of Peptide Chemistry
autoantibody epitopes, pin-bound peptides, modified ELISA, desmoglein 1
and 3, pemphigus
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Journal of Peptide Science
The mapping of linear B-cell epitope regions in desmoglein 1 and 3 proteins: Recognition of
immobilized peptides by pemphigus patients’ serum autoantibodies
Hajnalka Szabados; Szilvia Bısze; Pálma Silló; Sarolta Kárpáti; Ferenc Hudecz; Katalin Uray
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L.
University, Budapest 112, POBox 32, H-1518, HUNGARY
Department of Dermatology, Venereology and Dermato-Oncology, Semmelweis University,
Budapest, HUNGARY
Institute of Chemistry, Eötvös L. University, Budapest, HUNGARY
Abstract
Desmosomal transmembrane glycoproteins desmoglein 1 and desmoglein 3 are targets of lifethreatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus
foliaceus (PF). In these diseases pemphigus autoantibodies are produced against Dsg1 and
Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of
desmosomal cell-cell adhesion, and clinically, to the presence of blisters and erosions.
Identification, characterization and detailed analysis of the binding sites of autoantibodies
have an outstanding importance in understanding the immunopathology of the disease and
also in the design of novel diagnostics.
Here we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins
extracellular parts recognized by IgG-type serum autoantibodies of patients with PV and PF.
In our study overlapping pentadecapeptides were synthesized on hydroxypropylmethacrylate
pins based on the results of in silico predictions. To detect the interaction between the serum
autoantibodies and the immobilized synthetic peptides, modified ELISA (Enzyme Linked
Immunosorbent Assay) was performed with pin-attached peptides testing the serum samples
of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110,
aa196-220, aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and four
possible epitope regions (aa64-78, aa330-344, aa375-399, aa446-460) within the Dsg3 protein
sequence using these methods. Our data showed that serum autoantibodies of patients,
previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear
epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in
modified ELISAs.
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1. Introduction
Desmoglein (Dsg), a desmosomal Ca2+-dependent transmembrane glycoprotein, belongs to
the cadherin superfamily of cell-cell adhesion molecules with four isotypes: Dsg1, Dsg2,
Dsg3 and Dsg4 [1, 2]. These adhesion units specialize in conferring epidermal keratinocyte
cohesion and are linked to intercellular molecules of the desmosomal plaque, which in turn
interact with components of the cytoskeleton [3]. These glycoproteins are involved in the
development of autoimmune diseases. Dsg1 (1049 aa) and Dsg3 (999 aa) are targets of
Pemphigus vulgaris (PV), while Dsg1 is target of Pemphigus foliaceus (PF) also. These, and
especially PV, are life-threatening, autoimmune blistering disorders that affect the skin and
mucous membranes [4, 5].
There is no common theory for the pathomechanism of these diseases yet, however, it is
known that pemphigus autoantibodies are expressed against Dsg1 and Dsg3 proteins and bind
to them leading to a loss of desmosomal cell-cell adhesion, and clinically, to the presence of
blisters and erosions [6-10].
Although there are several diagnostic tools for autoimmune blistering diseases using monkey
esophagus and recombinant proteins, identification, characterization and detailed analysis of
the binding sites of autoantibodies has an outstanding importance in understanding the
immunopathology and development as well as in the design of novel diagnostics [11, 12].
Antibodies recognize parts of the protein antigen (antigenic determinants or epitopes) which
are created by the primary sequence of residues in the protein (called linear
epitopes/determinants) and/or by the secondary, tertiary or quaternary structure of the
molecule (called conformational epitopes/determinants) [13, 14]. Antigenic determinants are
usually limited to those portions of the antigen that are accessible to antibodies. Generally
these are small, 4-8 amino acid long residues frequently localized in or near to β-turns or loop
regions of a protein [15].
Antigenic determinants can be localized in theoretical ways using in silico secondary structure
prediction or 3D homology modeling of proteins [16], and experimental ways using domainswapped and point mutated recombinant proteins [17], synthetic protein fragments [18], X-ray
crystallography and NMR spectroscopy [19]. The use of small synthetic peptides of target
proteins can be an alternative way to identify epitope regions because of the sufficient
similarity to the native antigen which allows the binding of antibodies. This is a rapid,
practical, and cost-effective method for linear epitope region identification [20, 21]. B-cell
epitope mapping using a series of pin-attached overlapping synthetic peptides can be a very
efficient way to identify linear antigenic determinants recognized by serum antibodies or a
protein specific antibody in an immunoassay [22]. This strategy has successfully been used in
several studies [23-27] to identify antigenic epitope regions of viral, bacterial and other target
proteins.
Attempts to localize potentially antigenic regions within the desmoglein proteins have been
made previously with inconsistent results. Lucchese et al. have found a 12 amino acid long
peptide (aa49-60) on the extreme N-terminal region of the Dsg3 protein which was recognized
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by the sera of PV and PF patients, using a small number of synthetic peptides (Dsg1 aa49-60,
Dsg3 aa36-44, Dsg3 aa49-60, Dsg3 aa190-204, Dsg3 aa373-380, Dsg3 aa518-525) based on
the search of the least redundant peptide sequences [16]. In the domain swapping experiments
of Kawasaki et al. 87-194 residue long protein fragments were swapped between Dsg1 and
Dsg3 proteins. These studies showed the importance of the 1-88 [28] or 1-162 [17] region of
desmogleins. The same group performed point-mutations as well on human Dsg3 protein, and
found two short sequences (aa74-78 and aa102-105, recognized by Kawasaki’s mouse
monoclonal antibody) of which the amino acid residues were responsible for antibody
recognition [17]. Hacker-Foegen et al. identified a conformational epitope in the first 87 Nterminal residues of the Dsg1 protein using domain-swapped proteins and smaller
recombinant protein fragments [29]. Bhol et al. identified two antigenic protein fragments
BOS1 (aa50-79) and BOS6 (aa200-229) from Dgs3 protein sequence [18] with the usage of
12 synthetic 30mer peptides nearly covering the Dsg3 aa50-468 region. Dworschak et al.
analyzed 254 14mer peptides from the aa50-566 region of the Dsg3 protein and selected 5
larger peptides about 17-33 amino acid length and 2 original 14mer for further synthesis and
studies. These peptides were the following: aa50-78, aa243-255, aa273-293, aa304-330,
aa333-365, aa371-383, aa431-452. The N-termini of the peptides were biotinylated and
coupled to streptavidine containing ELISA plates. They identified the aa333-365 region as
major antigenic site and did not find any difference between normal human sera and PV
patient sera using the other peptides. [30].
The aim of the present study is to systematically localize linear B-cell epitope regions of the
extracellular parts of Dsg1 (aa50-550) and Dsg3 (aa50-625) proteins recognized by IgG-type
serum autoantibodies of patients with Pemphigus vulgaris and Pemphigus foliaceus using
theoretical and experimental methods as well. Therefore we have performed Chou-Fasman
secondary structure prediction studies [31] and Eisenberg hydrophobicity predictions [32] on
the extracellular part of Dsg1 and Dsg3 proteins. These methods have successfully been used
in several studies to identify hydrophilic β-turn segments of proteins [23, 24, 33]. For binding
studies, based on the results of predictions, overlapping pentadecapeptides were synthesized
on hydroxypropylmethacrylate pins based on the results of predictions. Finally, a modified
ELISA procedure has been optimized and ELISA experiments with eleven sera of ten patients
and with four healthy donors’ sera were performed on the pin-bound peptides.
We have defined five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340,
and aa486-520) within the Dsg1 protein sequence and four possible epitope regions (aa64-78,
aa330-344, aa375-399, aa446-460) within the Dsg3 protein sequence using these methods.
The defined epitope regions were subjected to homology search.
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2. Materials and methods
2.1. Chemicals and Reagents
Multipin peptide synthesis using Fmoc/tBu strategy
Multipin systems were obtained from Mimotopes (Clayton, Victoria, Australia). Fmoc
protected amino acid residues (Fmoc-AA) were purchased from Iris Biotech (Marktredwitz,
Germany). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N′-diisopropylcarbodiimide (DIC),
thioanisole, 1,2-ethanedithiol (EDT), phenol, piperidine, 1-hydroxybenzotriazole (HOBt),
N,N-diisopropylethylamine (DIEA) and trifluoroacetic acid (TFA) were purchased from
Sigma-Aldrich (Budapest, Hungary). N,N-dimethylformamide (DMF), methanol and acetic
anhydride (Ac2O) were obtained from Molar (Budapest, Hungary). N-methyl-2-pyrrolidone
(NMP) was purchased from Merck (Budapest, Hungary).
ELISA
Phosphate buffered saline (0.1M PBS, pH=7.4) was prepared by dissolving 150 mmole NaCl,
8 mmole Na2HPO4×2H2O, 2.7 mmole KCl and 15 mmole KH2PO4 (products of SigmaAldrich, Budapest, Hungary) in 1000 mL of deionized water. Gelatin, Tween-20, ophenylenediamine/H2O2 (SigmaFast), 2-mercaptoethanol and sodium dodecyl sulfate (SDS)
were also purchased from Sigma-Aldrich (Budapest, Hungary). Rabbit anti-human γimmunoglobulin conjugated to horseradish peroxidase (IgG/HRP) secondary antibody was
obtained from Dako (Glostrup, Denmark). 96-well microtest plates were the products of
Sarstedt (Nümbrecht, Germany).
2.2. B-cell epitope prediction
The human Dsg1 and Dsg3 sequences used for prediction were obtained from the protein
sequence database [34, 35] (http://www.ncbi.nlm.nih.gov/protein [last accessed June 2012]).
As antibody epitopes are located mainly on the hydrophilic surface of proteins, and many of
them at or near β-turn structures, we have predicted β-turns within the sequences of proteins
Dsg1 and Dsg3 using the Chou-Fasman secondary structure prediction method [31] and
performed Eisenberg hydrophobicity prediction [32]. In both cases, windows of seven amino
acids were used in MS Excel applying their original matrices. Segments with high probability
of β-turn secondary structure (Pβ-turn>1) and low probability of hydrophobicity
(Phydrophobicity<0) were selected. We have supported our results using the PredictProtein
website [36] (http://www.predictprotein.org/ [last accessed June 2012]).
Geno3D (http://geno3d-pbil.ibcp.fr [last accessed June 2012]), an automatic web server for
protein molecular modelling was used to obtain 3D model of human Dsg1 and Dsg3 proteins,
based on a cell adhesion (pdb3q2wA-0) and a structural protein (pdb1q55A-0) template [37].
2.3. Multipin peptide synthesis on a Mimotopes block
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Forty-five Dsg1 and forty-seven Dsg3 acetyl-pentadecapeptides overlapping in five amino
acid residues were synthesized in duplicates on Mimotopes’s hydroxypropylmethacrylate
non-cleavable pins (Figure 1, the gear contains the functional sites, nominal loading: 66 nmol)
with Fmoc/tBu chemistry according to Geysen’s method [22]. We used tBu (Thr, Ser, Tyr),
OtBu (Asp, Glu), Trt (His, Gln, Asn), Pbf (Arg), Acm (Cys) and Boc (Lys, Trp) as side chain
protecting groups. The Fmoc α-amino protecting group was removed with 2% piperidine, 2%
DBU/DMF (v/v) in 20 minutes. Then the pins were washed four times with DMF and twice
with methanol. Coupling was performed with 100 equivalent Fmoc-AA/DIC/HOBt 1:1:1
(n/n/n) in NMP for 2×60 min and monitored with bromophenol blue (0.1mM/DMF) added to
the coupling mixture [38]. At the end, the N-termini of the peptides were acetylated with
Ac2O/DIEA/DMF 5:1:50 (v/v/v) for 90 min, then after washing the pins the side chain
protecting groups (except Acm) were removed with TFA/thioanisole/phenol/water/EDT
82.5:5:5:5:2.5 (v/v/v/v/v) for 150 min, but the unprotected peptides remained covalently
attached to the pins.
2.4. Amino Acid Analysis
During and after the completions of the synthesis three-three pins were chosen as test
samples. Amino acid analysis was performed on Sykam Amino Acid S433H analyser
(Eresing, Germany) equipped with an ion exchange separation column and postcolumn
derivatization. Prior to analysis the gear was removed from the polyethylene pin (or rod,
Figure 1) and chopped; the gear-bound peptides were hydrolyzed with 6 M HCl in sealed and
evacuated tubes at 110°C for 24 h. For post-column derivatization the ninhydrin-method was
used. Amino acid analysis data are summarized in Table 1.
2.5. Human serum samples
Patients have been characterized as Dsg1 or Dsg3 positive according to the results of
Mesacup Dsg-1 (MB-RG7680EC-D) and Dsg-3 (MB-RG7685EC-D) ELISAs (MBL CO.,
LTD, Naka-ku, Nagoya, Japan) [4, 39, 40, 41]. Disease types have been identified with direct
and indirect immunofluorescence techniques (Department of Dermato-Venereology and Skin
Oncology, Semmelweis University). Characteristics of the serum samples are summarized in
Table 2. Eleven sera of ten patients of ages 23-89 (6♂, 4♀) were collected. Serum samples of
patients with mucosal PV (3 Dsg3 positive cases), sera of patients with PF (5 Dsg1 positive
cases), samples of patients with mucocutan PV (3 samples of both Dsg1 and Dsg3 positivity),
and four serum samples of healthy individuals were tested. Blood samples were centrifuged
on 4000 rpm for 15 minutes than the sera were collected and stored at -20°C before ELISA.
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2.6. Immunoserological epitope mapping using a modified ELISA with pin-bound peptides
To detect the interaction between the serum autoantibodies and the synthetic peptides,
modified ELISA (Enzyme Linked Immunosorbent Assay) was performed with pin-attached
peptides. First, the pins were incubated in 0.01M PBS (pH=7.4) containing 0.5% gelatin for
60 min at room temperature for saturation of non-specific binding sites and then washed twice
with 0.01M PBS. The serum samples were added to the ELISA plates in a dilution of 1:500
(diluted in 0.01M PBS containing 0.5% gelatin and 0.05% Tween-20). After overnight
incubation at room temperature, the pins were washed again for 5×5 min with 0.01M PBS.
Finally, the pins were incubated with rabbit anti-human IgG/HRP secondary antibody in a
dilution of 1:2000 (diluted in 0.01M PBS containing 0.5% gelatin and 0.05% Tween-20) for
60 min at room temperature. After washing with 0.01M PBS (5×5 min) the color reaction was
developed with o-phenylenediamine/H2O2 dissolved in deionized water and measured on
iEMS MF ELISA reader (Labsystem, Helsinki, Finland) at 492 and 620 nm 10 minutes later.
After the ELISA, the pins were regenerated by incubation in 0.1 M phosphate buffer, with 1%
(w/v) sodium dodecyl sulphate (SDS) pH 7.2 and 0.1% 2-mercaptoethanol. This was
performed at 60 °C in a sonicator bath (ULTRASONIK 3QT, 300ULTRASONIKA, 11,5 kW,
Pro-Analitika, Budapest, Hungary), for 5 minutes, twice. The pins were rinsed for 30 s in
MilliQ purified water pre-heated to 60 °C, followed by another rinse for at least 30 min in
purified water at an initial temperature of 60 °C, on a shaker table. To prepare the pins for
subsequent tests, they were immersed in methanol for 15 s and then air dried.
2.7. Homology search
We have compared the sequence of epitope regions of human Dsg1 (aa86-110, aa196-220,
aa226-250, aa326-340, aa486-520) and Dsg3 (aa64-78, aa330-344, aa375-399, aa446-460)
with that of proteins present in the non-redundant protein database by BLASTP 2.2.25+
program and used the following parameters according to the reference: Expect = 10; Matrix:
PAM30; Gap cost Existence: 9 Extension: 1; Word size = 2; filter: off; composition based
statistics: off [42] (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi [last accessed June 2012]).
For further analysis of the determined regions we used the Peptide Match Program
(http://www.pir.georgetown.edu/pirwww [last accessed June 2012]) to search for identical
peptide sequences (100% Blast) in the human proteome.
The Immune Epitope Database and Analysis Resource (http://www.immuneepitope.org [last
accessed June 2012]) have been also used with 70% minimum Blast to search for homologous
motifs in known epitopes of other proteins.
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3. Results and Discussion
3.1. B-cell epitope prediction
As antibody epitope regions are usually found at or near to hydrophilic β-turn segments of
proteins, 5 and 8 regions with high probability of β-turn secondary structure (Pβ-turn>1) and
low probability of hydrophobicity (Phydrophobicity<0) were selected within the extracellular part
of Dsg1 (aa50-550) and Dsg3 (aa50-625) proteins, respectively. The selection is based on the
results of Chou-Fasman secondary structure prediction, Eisenberg hydrophobicity prediction
and the results of the prediction provided by the PredictProtein website. These regions cover
the following sequences: aa56-110, aa126-305, aa306-370, aa381-405, aa406-550 from Dsg1,
and aa54-98, aa100-141, aa150-194, aa200-364, aa375-399, aa416-460, aa471-565, aa571625 from Dsg3 protein (Figure 2.).
3.2. Non-cleavable Multipin peptide synthesis
For immunoserological examination of the predicted regions, multipin systems with two sets
of overlapping pentadecapeptides covering regions I-V (Dsg1) and I-VIII (Dsg3) were
synthesized with solid phase Fmoc/tBu chemistry. Forty-five Dsg1 and forty-seven Dsg3
pentadecapeptides overlapping in five amino acid residues were prepared in duplicates on
hydroxypropylmethacrylate pins. Peptides used for B-cell epitope mapping were acetylated at
the N-terminus to avoid non-specific ionic interactions. The side chain protecting groups of
the amino acids were removed at the end of the synthesis, except Acm from Cys (mimicking
disulfide bridges in the protein). During and after the completions of the synthesis three-three
pins were chosen as test samples for amino acid analysis. Amino acid analysis of peptides
synthesized on pins yielded the ratios summarized in Table 1 (the number in parentheses is
the theoretical value) and confirmed that the synthesis of pin-attached peptides was
successful. It should be noted that these analyses were carried out on the side-chain protected
peptides and this may affect the measured values. The amino acid analysis also confirmed that
the amount of peptide on each tested pin was similar, approximately 40 nmol/pin (detailed
data not shown), which is a sufficient amount of pin-attached peptide for the majority of
antigen – antibody interactions.
3.3. Immunoserological epitope mapping using pin-attached synthetic peptides
The pin-attached overlapping peptides were tested with ELISA optimized by our laboratory.
The peptides were synthesized on non-cleveable funcionalized gears attached to polyethylene
pins. These pins are arranged in a modular unit which complements the 96-well format of a
microtiter plate. The pins with the gears are of such configuration that they fit into microtiter
wells. This feature greatly simplifies the handling of the pins. All pins with peptides are
contacted with serum samples and autoantibodies from the serum were bound to certain
peptides. After the pins were taken out of the serum sample and washed, the autoantibodies
remained bound to the peptides and were tested with enzyme-labeled, human IgG specific
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antibody conjugates for the presence of the peptide bound autoantibody. These IgG type Dsg
protein specific autoantibodies were detected from the sera of ten patients (eleven samples;
mucosal PV, mucocutan PV, PF) and four healthy donors. The serum antibodies of the
healthy individuals did not show any specific recognition in the tests. Therefore, the following
recognition criteria were chosen as positive recognition: patients’ measured OD ≥ mean OD +
3× SD value of healthy donors.
The results of Dsg1 and Dsg3 protein epitope mapping are summarized in Table 3. The fortyfive synthetic pin-bound pentadecapeptides covered 83% of the extracellular region of Dsg1
protein. From forty-five pentadecapeptides thirteen were recognized (29%) by the majority of
the autoantibodies of both Dsg1 positive and Dsg3 positive sera, twelve were recognized
(27%) only by the majority of the autoantibodies of Dsg3 positive sera, while twenty did not
have any recognition (44%).
The forty-seven pentadecapeptides studied from the Dsg3 protein sequence also covered 83%
of the extracellular region of the protein. Among the forty-seven peptides eight were
recognized (17%) by the majority of the serum autoantibodies of the patients, six were
recognized (13%) only by the majority of the autoantibodies of Dsg3 positive sera and one
were recognized (2%) only by the majority of the autoantibodies of Dsg1 positive sera. The
remaining thirty-two peptides did not have any recognition (68%).
According to the immunoserological analysis of the synthetic overlapping peptides we have
determined five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340,
aa486-520) within the Dsg1 protein sequence and four of those (aa64-78, aa330-344, aa375399, aa446-460) within the Dsg3 protein sequence (Figure 3) with high recognition.
Recognition of these epitope regions by autoantibodies from serum samples are summarized
in Table 4. To exemplify these data, detailed analysis of the two peptides within the Dsg1
aa196-220 region (Figure 4a,b) and of peptide Dsg3 aa64-78 (Figure 4c) is presented in
Figure 4. Our results with the highlighted samples (#11185, #11524, #11873, #11096,
#11696, #11608, #11689) show good matches with the clinical characteristics. Serum samples
#11096 and #11696 are from the same patient collected at two different time points. In case of
the latter sample (#11696) lower values were measured with the Mesacup Dsg-3 (MBRG7685EC-D) ELISA, corresponding to the lower recognition of individual peptides. Serum
autoantibodies of the Dsg1 positive samples #11542, #11190 recognized epitope regions from
the Dsg3 protein as well, while autoantibodies from Dsg3 positive samples #10915, #11205
showed recognition with not only Dsg3, but also Dsg1 peptides.
3.4. Homology studies of the identified epitope regions
The results of the homology search of the identified epitope regions are presented in Table 5.
As we expected, immunologically relevant hits showed homology mostly with proven or
predicted Dsg1, Dsg3, Dsg2, Dsg4 proteins, although some hypothetical and bacterial protein
hits were found as well.
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Further analysis of the identified regions we used the Peptide Match Program to search for
identical peptide sequences (100% Blast) in the human proteome. No match was found for
any of the determined regions in the database.
The Immune Epitope Database and Analysis Resource have been also used with 70%
minimum Blast to search for homologous motifs in known epitopes of other proteins. From
the possible antigenic regions of Dsg1 protein sequence aa196-220 showed homology with a
human transaldolase 1 epitope (aa231-245). We have to consider this in the further
examination of that sequence. Other identified regions did not show any homology with
epitopes from the human proteome.
4. Conclusion
Mapping of B-cell epitopes (antigenic determinants, antibody combining sites) of a protein is
the essential part of the design of synthetic diagnostics. These epitopes can be mapped using
numerous methods: they can be predicted from the amino acid sequence of a protein. They
can be assessed using synthetic peptides. Short synthetic peptides are suitable tools for
locating the epitope regions of larger proteins recognized by autoantibodies. In this paper we
presented that the multipin synthesis of overlapping peptides combined with modified ELISA
is a suitable method to find continuous linear epitope regions of immunodominant proteins in
PV. Also, analytical information was given in this study as to the amounts of amino acids
present on the selected individual pins.
In our study with overlapping pentadecapeptides tested in modified ELISAs we identified
epitope regions in the extracellular region of the native Dsg1 and Dsg3 proteins. In contrast to
Hacker-Foegen’s study [29] we identified five linear epitope regions (aa86-110, aa196-220,
aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and demonstrated
that not only conformational, but linear epitope regions are spread in the entire extracellular
region of the protein as well. From the Dsg3 protein sequence four linear epitope regions
(aa64-78, aa330-344, aa375-399, aa446-460) were determined containing two new linear
epitope regions (aa375-399 and aa446-460). In contrast to Dworschak’s study [30] our results
demonstrated that we found a smaller antigenic site (aa330-344) than aa333-365 and in our
experiments aa64-78 and aa375-399 were recognized by the antibodies of the patients as well.
Our experiments showed that serum autoantibodies of patients, previously identified as Dsg1
and Dsg3 positive, are able to recognize sequences of both Dsg1 and Dsg3 proteins. This
result may be in accordance with the fact that more proteins will be affected in the progress of
these autoimmune blistering skin disorders, which can be explained with the high homology
of these proteins. The identified regions showed homology mostly with desmogleins,
although some hypothetical and bacterial protein hits were found as well. One possible
epitope region within the Dsg1 protein sequence (aa196-220) showed homology with another
epitope peptide from the human proteome. Further analysis of the possible epitope regions is
planned in order to locate the exact antibody epitopes of Dsg1 and Dsg3 proteins.
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Table 1. Amino acid composition of some peptides synthesized on pins. The number in
parentheses is the theoretical value. Gly values are not determined as the spacer moiety of the
gear include glycine and β-alanine [43].
Code
Sequence
Dsg1 391-405
TYVVTGNMGSNDKVG
Dsg1 396-405
GNMGSNDKVG
Dsg1 396-405
GNMGSNDKVG
Dsg1 462-470
GTILSIDDN
Dsg1 462-470
GTILSIDDN
Dsg3 280-294
ILSSELLRFQVTDLD
Dsg3 280-294
ILSSELLRFQVTDLD
Dsg3 330-344
KVVKALDYEQLQSVK
Amino acid analysis
(found [calculated])
D 3.07 [3]; T 1.87 [2]; S 1.12 [1];
V 2.76 [3]; Y 0.88 [1]; M 0.75
[1]; K 1.09 [1]
D 3.07 [3]; S 1.12 [1]; V 0.97 [1];
M 0.75 [1]; K 1.09 [1]
D 2.96 [3]; S 1.11 [1]; V 0.95 [1];
M 0.86 [1]; K 1.11 [1]
D 2.85 [3]; T 0.92 [1]; S 1.21 [1];
I 1.74 [2]; L 1.27 [1]
D 2.94 [3]; T 0.93 [1]; S 1.05 [1];
I 1.80 [2]; L 1.28 [1]
D 2.25 [2]; T 0.97 [1]; S 2.05 [2];
E 2.15 [2]; V 1.12 [1]; I 1.05 [1];
L 4.28 [4]; F 0.96 [1]; R 1.07 [1]
D 2.14 [2]; T 0.95 [1]; S 2.05 [2];
E 2.22 [2]; V 1.12 [1]; I 1.05 [1];
L 3.98 [4]; F 0.96 [1]; R 1.07 [1]
D 1.14 [1]; S 1.05 [1]; E 3.22 [3];
A 0.95 [1]; V 2.86 [3]; L 1.81 [2];
Y 0.97 [1]; K 3.17 [3]
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Table 2. Characteristics of the serum samples.
Serum
Dsg1 titer Dsg3 titer
number
(U/ml)1
(U/ml)1
10915
258
PV
Dsg3
11689
155
mucosal
11205
151
11608
195
89
PV
*
Dsg1,3
11096
39
63
mucocutan
11696*
102
27
11185
99,6
11542
328
PF
Dsg1
11190
273
11524
149
11873
102
1
results of Mesacup Dsg-1 (MB-RG7680EC-D) and Dsg-3 (MB-RG7685EC-D) ELISAs
*
serum samples collected from the same patient at two different time points
Disease
type
Positivity
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Table 3. Peptide recognition pattern by Dsg1 and Dsg3 positive serum samples.
Recognition
of Dsg3
positive
samples
(n=6)a
+
+
++
++
+
+
+++
++
+
+
++
++
+
++
++
+++
+
++
+
++
+++
++
++
++
++
-
Recognition
of Dsg1
Region
Dsg3 peptides
positive
Region
Dsg1 peptides
samples
(n=8)a
56
54
I.
AACREGEDNSKRNPI70
I.
FAKPCREGEDNSKRN68
+
66
64
KRNPIAKIHSDCAAN80
NSKRNPIAKITSDYQ78
76
90
74
88
DCAANQQVTYRISGV
TSDYQATQKITYRIS
86
84
RISGVGIDQPPYGIF100
TYRISGVGIDQPPFG98
++
96
100
PYGIFVINQKTGEIN110
II.
FVVDKNTGDINITAI114
126
114
II.
YCRALNSMGQDLERP140
IVDREETPSFLITCR128
136
150
127
141
DLERPLELRVRVLDI
CRALNAQGLDVEKPL
146
150
RVLDINDNPPVFSMA160
III.
INDNPPVFSQQIFMG164
156
170
160
174
VFSMATFAGQIEENS
QIFMGEIEENSASNS
166
170
IEENSNANTLVMILN180
SASNSLVMILNATDA184
176
180
VMILNATDADEPNNL190
NATDADEPNHLNSKI194
186
200
EPNNLNSKIAFKIIR200
IV.
SQEPAGTPMFLLSRN214
196
210
210
224
FKIIRQEPSDSPMFI
LLSRNTGEVRTLTNS
206
220
SPMFIINRNTGEIRT220
TLTNSLDREQASSYR234
216
230
230
244
GEIRTMNNFLDREQY
ASSYRLVVSGADKDG
226
240
DREQYGQYALAVRGS240
ADKDGEGLSTQCECN254
236
250
AVRGSDRDGGADGMS250
QCECNIKVKDVNDNF264
246
260
ADGMSAECECNIKIL260
VNDNFPMFRDSQYSA274
256
270
270
284
NIKILDVNDNIPYME
SQYSARIEENILSSE
266
280
IPYMEQSSYTIEIQE280
ILSSELLRFQVTDLD294
276
290
290
304
IEIQENTLNSNLLEI
VTDLDEEYTDNWLAV
286
300
NLLEIRVIDLDEEFS300
NWLAVYFFTSGNEGN314
291
310
RVIDLDEEFSANWMA305
GNEGNWFEIQTDPRT324
306
320
III.
VIFFISGNEGNWFEI320
TDPRTNEGILKVVKA334
316
330
330
344
NWFEIEMNERTNVGI
KVVKALDYEQLQSVK
+
326
340
340
354
TNVGILKVVKPLDYE
LQSVKLSIAVKNKAE
336
350
PLDYEAMQSLQLSIG350
KNKAEFHQSVISRYR364
+
346
360
375
389
QLSIGVRNKAEFHHS
V.
INVREGIAFRPASKT
+
356
385
EFHHSIMSQYKLKAS370
PASKTFTVQKGISSK399
+++
381
416
IV.
EGPVFRPGSKTYVVT395
VI.
TNKAASNVKYVMGRN430
391
426
TYVVTGNMGSNDKVG405
VMGRNDGGYLMIDSK440
+
406
420
436
450
V.
DFVATDLDTGRPSTT
MIDSKTAEIKFVKNM
416
446
RPSTTVRYVMGNNPA430
FVKNMNRDSTFIVNK460
426
440
471
485
GNNPADLLAVDSRTG
VII.
EYTGKTSTGTVYVRV
436
481
DSRTGKLTLKNKVTK450
VYVRVPDFNDNCPTA495
446
491
NKVTKEQYNMLGGKY460
NCPTAVLEKDAVCSS505
456
501
LGGKYQGTILSIDDN470
AVCSSSPSVVVSART515
466
480
511
525
SIDDNLQRTCTGTIN
VSARTLNNRYTGPYT
476
490
521
535
TGTININIQSFGNDD
TGPYTFALEDQPVKL
+
486
500
531
545
FGNDDRTNTEPNTKI
QPVKLPAVWSITTLN
496
541
PNTKITTNTGRQEST510
ITTLNATSALLRAQE555
+
506
551
RQESTSSTNYDTSTT520
LRAQEQIPPGVYHIS565
516
571
DTSTTSTDSSQVYSS530
VIII.
SQNNRCEMPRSLTLE585
526
540
581
595
QVYSSEPGNGAKDLL
SLTLEVCQCDNRGIC
536
550
591
605
AKDLLSDNVHFGPAG
NRGICGTSYPTTSPG
601
TTSPGTRYGRPHSGR615
611
PHSGRLGPAAIGLLL625
a
Criteria: patients’ measured OD ≥ mean OD + 3× sd value of controls. +++: positive recognition by n out of n patients, ++: positive
recognition by n-1 out of n patients, +: positive recognition by n-2 out of n patients.
Recognition
of Dsg1
positive
samples
(n=8)a
+
++
+
+
+++
++
++
+
++
+
++
+
+
-
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Recognition
of Dsg3
positive
samples
(n=6)a
+
++
+
+
+
++
++
++
+
+
+
+
+
+
-
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Table 4. Recognition of the identified epitope regions by the serum samples.
Dsg1+a
Dsg1,3+a
Dsg3+a
Protein Sequence
11542 11185 11190 11524 11873 11096
11696
11608 10915 11689 11205
86-110
+
+
+
+
+
+
+
+
+
196-220
+
+
+
+
+
+
+
+
+
+
Dsg1
226-250
+
+
+
+
+
+
+
+
+
+
326-340
+
+
+
+
+
+
+
+
+
+
486-520
+
+
+
+
+
+
+
+
+
11542 11185 11190 11524 11873 11096
11696
11608 10915 11689 11205
+
+
+
+
+
+
+
64-78
+
330-344
+
+
+
+
+
+
+
+
Dsg3
375-399
+
+
+
+
+
+
+
+
446-460
+
+
+
+
+
+
+
a
according to the results of Mesacup Dsg-1 (MB-RG7680EC-D) and Dsg-3 (MB-RG7685EC-D) ELISAs
+: recognition; -: no recognition (criteria: patients’ measured OD ≥ mean OD + 3× sd value of controls)
b
sequence of the determined epitope region
All sera were tested for both Dsg1 and Dsg3 derived pin-attached peptides. Highlighted areas show good matches with the
clinical characteristics.
b
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Table 5. Homology search results. All subsequences recognized by serum autoantibodies
possess high score hits to the Dsg1 (in case of Dsg1 subsequences) and Dsg3 (in case of Dsg3
subsequences) proteins first, and some other adhesion proteins, hypothetical and bacterial
proteins were found with lower score as well.
Dsg1 subsequences
86-110
196-220
226-250
326-340
486-520
Dsg3 subsequences
64-78
330-344
375-399
446-460
Description
Dsg1, other desmogleins, cadherin-8, some hypothetical proteins
Dsg1, other desmogleins, cadherins, some bacterial and virus proteins
Dsg1, Dsg4, some hypothetical and bacterial proteins
Dsg1, other desmogleins, desmocollin, cadherins
Dsg1, some hypothetical and bacterial proteins
Description
Dsg3, other desmogleins
Dsg3, other desmogleins
Dsg3, other desmogleins, some hypothetical proteins
Dsg3, some hypothetical and bacterial proteins
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Figure legends
Figure 1. The outlook of the Mimotopes non-cleavable pin.
Figure 2. Prediction profiles of Dsg1 (a) and Dsg3 (b) proteins generated by Chou-Fasman
secondary structure prediction and Eisenberg hydrophobicity prediction. Dark gray: ChouFasman β-turn prediction; light gray: Eisenberg hydrophobicity prediction; black: selected
regions for multipin peptide synthesis.
Figure 3. Localisation of experimentally determined epitope regions on 3D homology model
of human Dsg1 (a) and Dsg3 (b) proteins. Red ribbons: 8/8 (Dsg1+ patients) positive
recognition; blue ribbons: 7/8 (Dsg1+ patients) and 5/6 (Dsg3+ patients) positive recognition.
Figure 4. Binding of serum autoantibodies to pin-bound pentadecapeptides. Detailed
representation of modified ELISA results in case of peptides within Dsg1 aa 196-220 (a,b)
and peptide Dsg3 aa64-78 (c). D1-D4 indicate the binding of healthy control sera.
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Figure 1. The outlook of the Mimotopes non-cleavable pin.
24x33mm (300 x 300 DPI)
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Figure 2. Prediction profiles of Dsg1 (a) and Dsg3 (b) proteins generated by Chou-Fasman secondary
structure prediction and Eisenberg hydrophobicity prediction. Dark gray: Chou-Fasman β-turn prediction;
light gray: Eisenberg hydrophobicity prediction; black: selected regions for multipin peptide synthesis.
25x20mm (300 x 300 DPI)
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Figure 3. Localisation of experimentally determined epitope regions on 3D homology model of human Dsg1
(a) and Dsg3 (b) proteins. Red ribbons: 8/8 (Dsg1+ patients) positive recognition; blue ribbons: 7/8 (Dsg1+
patients) and 5/6 (Dsg3+ patients) positive recognition.
32x25mm (300 x 300 DPI)
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Figure 4. Binding of serum autoantibodies to pin-bound pentadecapeptides. Detailed representation of
modified ELISA results in case of peptides within Dsg1 aa 196-220 (a,b) and peptide Dsg3 aa64-78 (c). D1D4 indicate the binding of healthy control sera.
78x152mm (300 x 300 DPI)
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